Description The Epidermal Growth Factor Receptor-family tyrosine kinases EGFR and HER2 are overexpressed in a significant proportion of aggressive, treatment-refractory breast cancers. This includes HER2+ tumors that have acquired resistance to HER2-specific monoclonal antibodies and kinase inhibitors and Triple-Negative Breast Cancers (TNBCs), of which approximately 50% overexpress EGFR. Agents that selectively kill EGFR+ or HER2+ tumors through a novel mechanism of action could improve the survival of patients with these treatment-refractory cancers. Our team reported a new class of anticancer compounds, termed DDAs, with selective toxicity against EGFR+ and HER2+ cancers. Published and preliminary results demonstrate that DDAs kill patient-derived breast xenograft tumors without detectable toxicity to experimental animals. DDAs kill cancer cells by activating Death Receptors 4 and 5 (DR4/5), the receptors for the anticancer cytokine TRAIL. However, DDAs activate DR4/5 in a novel TRAIL-independent manner. DR4/5 activation by DDAs results from a combination of elevated DR5 expression, disulfide bond-dependent DR4/5 clustering, and localization of DR4/5 to the cell surface. These events occur specifically in EGFR or HER2 overexpressing cancer cells and result in DR4/5 activation of the Caspase 8-Caspase 3 pro-apoptotic cascade. Consistent with this in vitro selectivity, tumor studies demonstrate DDA-induced apoptosis of cancer cells without toxicity to adjacent normal tissues. This project will test the hypothesis that the protein disulfide isomerase AGR2 catalyzes/chaperones native disulfide bonding of EGFR, HER2, DR4, and DR5. We further hypothesize that DDAs inhibit AGR2 enzyme activity and that EGFR and HER2 strongly potentiate DDA actions by competing with DR4/5 for the limited pool of AGR2. Specific Aim 1 is to elucidate the mechanisms by which disulfide bonding within the extracellular domains of DR4/5 restrains their basal pro-apoptotic activity, and demonstrate that genetic or pharmacologic DR5 activation in cancer cells is sufficient to induce tumor regression. Specific Aim 2 is to investigate the mechanisms by which DDAs block AGR2 PDI activity and to demonstrate that DR4/5 activation mediates cancer cell apoptosis in response to ablating AGR2 activity. Specific Aim 3 is to establish the efficacy of DDAs against patient-derived breast tumors and verify that the mechanisms of DDA action in vivo match those identified in vitro. DDA efficacy will be compared with standard therapies in single agent and combination regimens. In summary, this exploratory project will investigate DDAs as a new class of anticancer agents, elucidate a novel AGR2/Death Receptor 4/5 cascade that mediates DDA actions, and establish DDA efficacy against patient-derived models of metastatic and drug-resistant HER2+ breast cancer.